Plasminogen activators are of great importance in treating occlusions of blood vessels by fibrin clots, as in acute myocardial infarction, pulmonary embolism, arterial thromboembolism and other clinical conditions.
Since about 1951 it has been known that human urine contains a proteolytic enzyme which by limited proteolytic activates plasminogen to plasmin, i.e., the enzyme which degrades fibrin to soluble polypeptides and thus causes the dissolution of the fibrin component of blood clots. This plasminogen activator first isolated from urine in 1951 was named urokinase. Later several different forms of urokinase were described, which all turned out to be related to the same precursor molecule. This zymogen "prourokinase" has been known since at least 1977. The primary structures of the two major forms of urokinase and the structure of prourokinase were elucidated, however, only in 1982 and 1984, respectively. Prourokinase has a single chain structure. Therefore it was designated scu-PA (single chain urinary plasminogen activator). This scu-PA consists of 411 amino acids, and in its naturally occurring form it is glycosylated at the amino acid in position 302 of the protein chain.
Several papers describe the production of the unglycosylated protein moiety of scu-PA by recombinant techniques, in Escherichia coli (hereinafter E. coli), which is designated as "recombinant scu-PA" (recombinant scu-PA; proposed INN "Saruplase"). In a randomized double-blind therapeutic trial on 401 patients, recombinant scu-PA proved to be more efficient in thrombolysis and better tolerated than conventional fibrinolytic agents (see, for example, The Lancet, Apr. 22, 1989, pp. 863 to 868).
As described in various publications, procaryotic organisms can be used to produce the unglycosylated plasminogen activator recombinant scu-PA. The processes known for the production of the recombinant scu-PA involve the expression of the scu-PA structural gene obtained from human tissues. Unfortunately, in these processes only very low expression rates are obtained. These low yields make impossible economically feasible large scale production of the desired product. For instance, Hibino et al., in Agric. Biol. Chem. 52:329-336 (1988), obtained an expression of only 2% from recombinant E. coli when calculated per amount of total bacterial protein. Other publications such as, for example, the European patent application published under number 00 92 182 A2 and Holmes et al., Biotechnol. 3:923-929 (1985) give no precise values of the expression rates obtained. Reproduction of these experiments in different bacterial strains, however, also has yielded recombinant scu-PA expression of less than 2% of the bacterial protein.
As in most cases of expressing eukaryotic proteins in bacteria, the recombinant scu-PA protein chain is produced by the transformed bacterial cell in the form of inclusion bodies consisting of denatured protein designated hereinafter as "intermediate protein". This intermediate protein, after isolation, must be renatured ("refolded") by chemical treatment to form the correct tertiary structure of recombinant scu-PA that is suitable for use as a thrombolytic agent. To determine the amount of recombinant scu-PA formed, the resulting product may be treated with plasmin to transform recombinant scu-PA to recombinant (unglycosylated) two chain urokinase ("rtcu-PA"), the enzymatic activity of which is then determined and may serve as a measure of the amount of recombinant scu-PA formed. Of course, it is also possible to determine the amount of intermediate protein formed or the amount of recombinant scu-PA directly, for example, by densitometric scanning of the total bacterial proteins after electrophoretic separation, and thereby determine the expression rate.
Despite a great deal of work which has been done in the art, there remains a need for means and techniques for expressing scu-PA at rates which facilitate economically feasible production of recombinant scu-PA.